The present invention relates to the sequencing of DNA and in particular to the selection of unique templates for DNA sequencing. The selection methods of this invention can be performed fully in vitro. The invention further relates to a kit which can be used when employing the selection method of this invention.
DNA sequencing is a widely used method which is indispensable within modem molecular biology. One factor limiting sequencing is the shortness of the sequence to be read using one primer. Present-day techniques make it possible to read about 1000 bases starting from the primer. In practice, the range to be read covers about 400 to 800 bases. When sequencing a long DNA the examined DNA has been divided up into smaller fragments and the fragments have been separately sequenced, or the so called xe2x80x9cDNA walkxe2x80x9d method has been used.
In addition, it is essential that only one template is used in a single sequencing reaction. In the known methods, the problem has been solved by transforming the sequencing vector comprising the examined DNA into a bacterial cell, usually into Escherichia coli, and by isolating single colonies from the culture plate.
1. In the xe2x80x9cDNA walkxe2x80x9d method, the examined DNA is inserted into a vector and the inserted DNA is sequenced as far as possible by a known primer located in the vector. Using the obtained sequence, a new primer is designed, on which the sequencing is further continued (Itakura et al 1984 and Sambrook et al, 1989). The method is repeated until the entire sequence under examination has been exposed. The method is time-consuming and costly. The result from the previous round is needed for the next round, and new unique primers are required for each round.
2. The dividing up of the DNA into smaller, overlapping fragments has been performed by a number of different methods:
DNA mapping using restriction enzymes and subcloning of the restriction products into sequencing vectors (Sambrook et al, 1989);
the so called xe2x80x9cshot gunxe2x80x9d sequencing method where the DNA is randomly divided up into fragments and cloned into sequencing vectors (Sulston et al, 1992), and
the so called exodeletion method where the DNA under examination is ligated into a sequencing vector and shortened by means of an exonuclease (Sambrook et al, 1989 and Ausubel et al, 1989).
All of the above methods are laborious and in each one, the DNA must be transformed into a bacterium (usually into E. coli) so as to produce a unique DNA template to be sequenced.
Furthermore, various kinds of methods based on DNA transposition have been disclosed for sequencing DNA. The majority of these methods are based on in vivo transposition (U.S. Pat. No. 4,716,105 and Strathmann et al 1991). The only known sequencing method (U.S. Pat. No. 5,728,551) exploiting in vitro transposition is based on using Ty1 integrase activity and even here, only the transposition reaction is carried out in vitro; the transposition products are transformed into Escherichia coli so as to obtain single sequencing templates. Related art is disclosed also in the U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,212,080, U.S. Pat. No. 5,645,991, and in Kasai et al (1992), Krishnan et al (1993), and Roach et al (1995).
It would be highly advantageous if the selection of single sequencing templates through a single cell culture step could be fully avoided. Even in the known, partly in vitro methods, the amplification products should be transformed into a bacterium and single colonies are to be picked in order to obtain unique templates for sequencing the DNA.
The present invention aims at eliminating the problems relating to the prior art and, therefore, it is an object of the present invention to provide a simple method for fragmenting larger DNA segments into pieces enabling the sequencing of whole segment by using one or a few sequencing primers.
It is another object of this invention to provide, fully in vitro, a unique sequencing template for a single DNA sequencing reaction.
According to one embodiment (later called embodiments A and B) of this invention the examined DNA is subjected to a DNA transposition reaction and to an amplification reaction. The amplification reaction is carried out in the presence of a first, fixed primer, hybridizing at a known position in the examined DNA or if the examined DNA is part of a vector for example, adjacent to the examined DNA, and a second, selective primer, hybridizing at the insertion site of a transposon. These reactions result in amplification products which can be separated on the basis of their size difference. By suitably selecting the amplification products, whose size difference is in the range of the reading capacity of the sequencing system used, normally in the range of 200 to 600 bp, overlapping DNA templates can be obtained for sequencing. The target DNA can range from a few base pairs up to 40 kilo base pairs. The only limiting factor for not choosing as target DNA even longer DNA segments is the inability of the present amplification reactions, such as PCR, to produce longer DNA segments.
According to another embodiment (later called embodiment C) of this invention a transposition reaction is carried out in the presence of the examined DNA and in the presence of a transposon or transposons which have different ends enabling the design of selective primers for both ends. The transposon ends can be part of the same molecule or separate molecules. The amplification reaction is carried out in the presence of the selective primers designed for both transposon ends. The amplification products are situated at random positions in the target DNA. To be able to cover the whole sequence of the target DNA, a sufficient number of the amplification products needs to be selected for sequencing. Very long DNA segments can be sequenced by selecting the templates according to this embodiment. The present-day amplification reactions, such as PCR, are not a limiting factor. One advantage is that no sequence data of the target DNA is needed, nor is any subcloning procedure necessary.
One object of this invention is to provide a kit which comprises a transposon recognizable by a transposase enzyme, the transposase enzyme, a fixed primer hybridizing at a known position in the examined DNA or if the examined DNA is part of a vector for example, adjacent to the examined DNA, and a selective primer including a sequence complementary to the transposon joining end.
One further object of this invention is to provide a kit which comprises a selective primer, a fixed primer, a transposon recognizable by a transposase enzyme, and the transposase enzyme, and wherein the transposon and the transposase enzyme are provided as a transposition complex.
One still further object of this invention is to provide a kit which comprises two different transposon ends recognizable by a transposase enzyme or transposase enzymes, the transposase enzyme or transposase enzymes, and selective primers including a sequence complementary to the transposon joining ends.